It has been recently found that in the process of differentiation from the undifferentiated hematopoietic stems cell to mature blood cells, a number of hematopoietic factors mutually interact at various levels to form a complicated hematopoietic network system. In addition, most of these hematopoietic factors are gene-cloned and at present time, some of the hematopoietic factors are mass-produced by means of genetic recombination technologies and their clinical applications have been developed. On the other hand, the undifferentiated hematopoietic stem cells exhibit a feature having capacity of self-renewal (proliferation) but growth factors that could act on the undifferentiated hematopoietic stem cells in the bone marrow have not been sufficiently clarified.
It is known that for proliferation of the hematopoietic stem cells in the bone marrow and differentiation to the mature cells, the bone marrow stromal cells play a central role and it is suggested that the humoral factors which the stromal cells secrete and/or cell-cell interactions take part in the proliferation of undifferentiated hematopoietic stem cells.
For example, it is known that a bone marrow stromal cell MC3T3-G2/PA-6(PA-6) established from the calvaria of a C57B1/6 neonatal mouse supports proliferation of murine pluripotent hematopoietic stem cells [Kodama H. et al: J. Cell Physiol., 112, 89 (1982)].
Recently, a ligand for c-kit protein on the surface of the hematopoietic stem cells is given attention as a factor taking part in the proliferation of the stem cell and researches for clarifying the true function have been extensively conducted and as the result, three groups have succeeded in the gene cloning to report SCF [stem cell factor; K. M. Zsebo et al: Cell, 63, 195-201 (1990)], MGF [mast cell growth factor; D. E. Williams et al: Cell, 63, 167-174 (1990) and KL [c-kit ligand; Huang et al: Cell, 63, 225-233 (1990)].
At present time, by using c-kit ligand mass-produced by means of a genetic recombination technology, analysis of the function has been developed and it has been clarified from researches by now that this factor supports proliferation of the hematopoietic stem cells having been differentiated in some extent [Hayashi et at.: Int. J. Hematology, Supple. No. 1, p198 (1990)]. Therefore, it is considered that besides this protein, another factor acting on the undifferentiated stem cell should exist in the bone marrow.
A hematopoietic factor supporting the proliferation of undifferentiated stem cells will be a useful medicine which is used as a remedy for suppression of the bone marrow, for example, recovery of suppression of the bone marrow in chemotherapy for cancer or bone marrow transplantation or a remedy for bone marrow malfunction such as aplastic anemia and myelodysplasia syndrome and furthermore, used for bone marrow transplantation as an in vitro growth agent for the peripheral blood stem cells and the bone marrow stem cell.
The purpose of the present invention is to provide a new protein having growth activity for undifferentiated hematopoietic stem cells and the hematopoietic stem cells growth agent containing said protein as an active ingredient and used for curing for suppression of the bone marrow, or curing for incompetence of the bone marrow or an in vitro proliferation for the peripheral blood stem cells and the bone marrow stem cells.
As it is known that the bone marrow stromal cells are constituted of the fibroblast/the preadipocyte, the endothelial cell and the macrophage and produces such hematopoietic factors as interleukin 1, interleukin 6, interleukin 7, interleukin 8, interleukin 11, c-kit ligand, LIF (leukemia inhibitory factor), GM-CSF, G-CSF and M-CSF, the present inventors thought that a growth factor to the undifferentiated hematopoietic stem cells exists in the human fibroblast cell-conditioned medium.
It is known that the hematopoietic cells obtained from the bone marrow of a mouse in which a large amount of 5-fluorouracil (5FU) had been administered are called pre spleen colony forming &Its (pre CFU-S) and are the undifferentiated hematopoietic stem cells with high capacity of self-renewal.
The present inventors have extensively studied to aim separation of a new physiologically active protein from a culture medium of the fibroblast by using growth activity to the 5-FU resistant murinebone marrow cells as a target. As the result, a new physiologically active protein having a growth activity for the undifferentiated hematopoietic stem cells has been isolated to complete the present invention.